Human-type anti-blood coagulation factor VIII antibody

a technology human anti-fviii, which is applied in the field of can solve the problems of ineffective supplemental treatment of blood coagulation factor, difficult hemostatic management of hemophilia patients, and the inability to completely humanize the anti-fviii inhibitor antibody, and the antibody preparation of a complete human anti-fviii inhibitor antibody has not been successful

Inactive Publication Date: 2007-05-08
JURIDICAL FOUND THE CHEMO SERO THERAPEUTIC RES INST
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AI Technical Summary

Problems solved by technology

This antibody, called “anti-FVIII inhibitor antibody”, makes supplemental treatment with the blood coagulation factors ineffective and hemostatic management of the hemophilia patients extremely difficult.
A completely human anti-FVIII inhibitor antibody, however, has scarcely been prepared with success although it has been attempted over more than ten years in many laboratories with the hybridoma technique (human / mouse or human / human hybridoma technique) or the EB transformation technique, using lymphocytes from patients having anti-FVIII inhibitor antibodies.
nts. Moreover, although the clones obtained in these reports bound to FVIII as shown by ELISA, it was not proved whether the clones inhibited the coagulation activity of F
Accordingly, a human anti-FVIII antibody with the inhibitor activity has not yet successfully been isolated, hence failing to provide sufficient information.
The reason why a human anti-FVIII antibody with the inhibitor activity could not successfully be isolated with the antibody display phage technology was presumed to be that genes of VL chain of antibody fragments were not derived from patients and that there might be some problems in the screening procedure.

Method used

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  • Human-type anti-blood coagulation factor VIII antibody
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  • Human-type anti-blood coagulation factor VIII antibody

Examples

Experimental program
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Effect test

example 1

Construction of Phage Library from Hemophilia Patient:

[0058]A phage library was constructed with reference to the method reported by J. D. Marks et al. (J. Mol. Biol., 222: 581–597, 1991).

[0059]Lymphocytes were separated with Ficoll from peripheral blood (28 ml) from a patient having the anti-FVIII inhibitor antibody, washed thoroughly with PBS, and treated with ISOGEN (Nippon Gene Co., Ltd.) to prepare a total RNA. The obtained total RNA was divided into three portions, each of which was reverse-transcribed with p primers specific for the constant regions of human IgG, K chain or λ chain with First Strand cDNA Synthesis Kit (Pharmacia bio tec) to prepare cDNAs for respective constant regions. The obtained cDNAs were then used as a template for amplifying antibody V region genes by polymerase chain reaction (PCR) using primers specific for gene families as a combination of VH+JH, VK+JK, and Vλ+Jλ, as reported by Marks et al.

[0060]VH genes and VK genes, and VH genes and Vλ genes, res...

example 2

Screening:

[0061]Factor VIII (Cross Eight M; 10 units / mL) dissolved in a screening buffer (4% skim milk, 0.5% bovine serum albumin, 50 μg / mL mouse IgG, 0.1% Tween 20, TBS; 500 μL) and either 0.5 μg / mL of anti-Factor VIII monoclonal antibodies conjugated with biotin FVIII:C14-182 (mouse; recognizing Factor VIII L chain) or FVIII:C14-282 (mouse; recognizing FVIII H chain A2 domain) dissolved in a screening buffer (500 μL) were mixed in a plastic tube (FALCON 2058) to react at room temperature for 30 minutes. Thereto was added the phage library displaying human antibody derived from patients (a solution of phage displaying single-chain antibodies; 1 mL) to react at room temperature for 1 hour and then at 4° C. overnight.

[0062]Avidin bound magnetic particles (Perseptive; 100 μL) treated with 2% skim milk / 0.05% Tween 20 / TBS were added to the above mixture of anti-FVIII monoclonal antibody conjugated with biotin / FVIII (1 mL) and the mixture was gently mixed upside-down for 15 minutes. The ...

example 3

Expression and Recovery of scFv:

[0064]A soluble scFv was expressed with E. coli HB2151, recovered from E. coli periplasmic fraction and crudely purified. Affinity purification was performed with RAPAS Purification Module (Pharmacia Biotech) when further purification was needed.

[0065]Purity of the purified scFv protein was confirmed by SDS-polyacrylamide gel electrophoresis and Western blotting targeted to E-Tag epitope at the C-terminal of the scFv protein. A protein concentration of the purified scFv protein was determined with Protein Assay kit (BIO-RAD).

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Abstract

The present invention provides a human antibody against blood coagulation factor VIII (hereinafter also referred to as “FVIII”) and an antibody fragment that binds to human FVIII and specifically inhibits the coagulation activity of human FVIII. ScFv display phage libraries, prepared by using scFv DNAs constructed by random combinations of immunoglobulin VH chain genes and VL chain genes from lymphocytes from hemophilia A patients, is reacted with FVIII immobilized to a solid phase via anti-FVIII monoclonal antibody, and scFv clones capable of binding to FVIII are cloned to reveal VH and VL chains of FVIII-specific antibody.

Description

[0001]This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT / JP02 / 05783 which has an International filing date of Jun. 11, 2002, which designated the United States of America.TECHNICAL FIELD OF THE INVENTION[0002]The present invention relates to a provision of a human inhibitor antibody against blood coagulation factor VIII (hereinafter also referred to as “FVIII”) and an antibody fragment that bind to human FVIII and thereby specifically inhibits the coagulating activity of FVIII.BACKGROUND OF THE INVENTION[0003]Hemophilia A is a genetic disease where disorder or deficiency of FVIII leads to reduction of the blood coagulation activity, resulting in bleeding in the bowels or at the joint, etc. For treatment of patients suffering from hemophilia A, supplemental treatment of FVIII has been carried out. In about 10% (8 to 15%) out of the patients who received supplemental treatment, however, induction of an antibody to FVIII has been obser...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07H21/04A61K38/00C07K7/06C07K7/08C07K14/755C07K16/36C12P21/08G01N33/86
CPCC07K7/06C07K7/08C07K14/755C07K16/36G01N33/86A61K38/00C07K2317/21C07K2317/622A61P7/02C12N15/11
Inventor NAKASHIMA, TOSHIHIROYUGUCHI, MASATO
Owner JURIDICAL FOUND THE CHEMO SERO THERAPEUTIC RES INST
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